This invention relates to fatty acid metabolism, in particular to fatty acid desaturases.
Polyunsaturated fatty acids are important as structural components of membrane glycerolipids and as precursors of families of signaling molecules including prostaglandins, thromboxanes, and leukotrienes (Needleman et al., Annu. Rev. Biochem. 55:69-102, 1986; Smith and Borgeat, in Biochemistry of Lipids and Membranes, eds. Vance and Vance, Benjamin/Cummings, Menlo Park, Calif., 1986, pp. 325-360).
The principal fatty acid precursors of these signaling molecules are arachidonic acid (Δ5,8,11, 14-20:4), providing an ω-6 substrate that is responsible for the major synthesis of these compounds, and eicosapentanenoic acid (Δ5,8,11,14,17-20:5), an ω-3 substrate that is responsible for the parallel synthesis of many elcosanoids having an additional double bond. An important class of enzymes involved in the synthesis of polyunsaturated fatty acids is the fatty acid desaturases, which catalyze the introduction of double bonds into the hydrocarbon chain.
In vertebrates, desaturases are known to act at the Δ4, 5, 6, 8 and 9 positions (Holloway, In: The Enzymes, ed. Boyer, Academic Press, New York, vol. 16, 1983, pp. 63-83). The 18:0-CoA Δ9 desaturase from rat liver has been characterized biochemically (Strittmatter et al., Proc. Natl. Acad. Sci. USA 71:4565-4569, 1974; Thiede et al., J. Biol. Chem. 260:14459-14463, 1985), and the corresponding gene has been cloned (Thiede et al., J. Biol. Chem. 261:13230-13235, 1986). However, the remaining four enzymes have remained recalcitrant to purification and genes that encode them have not been isolated. Based on available information, and by analogy to the 18:0-CoA desaturase, it is likely that the remaining four enzymes are integral membrane proteins that require other membrane components (cytochrome b5 and NADH:cytochrome b5 reductase) for activity (Strittmatter et al., Proc. Natl. Acad. Sci. USA 71:4565-4569, 1974), and it is these features that have limited progress in studying the biochemistry and molecular genetics of these important synthetic reactions.
Biochemical studies of membrane-bound fatty acid desaturases in plants have proven equally difficult, and only one enzyme has been purified to homogeneity (Schmidt et al., Plant Mol. Biol. 26:631-642, 1994). Higher plants produce many different unsaturated fatty acids (Hilditch and Williams, The Chemical Constituents of Natural Fats, Chapman and Hall, London, 4th Ed., 1964), but in membrane lipids the major locations for double bonds are at the Δ9, 12 and 15 (ω-3) positions of 18-carbon acyl chains and the corresponding Δ7, 10 and 13 (ω-3) positions of 16-carbon chains (Browse and Somerville, Ann. Rev. Plant Physiol. Plant Mol. Biol. 42:467-5069, 1991).